Process for Preparation of a Grifola frondosa Polysaccharide F2 and Its Hypoglycemic Activity

ABSTRACT

A  Grifola frondosa  polysaccharide F2 with hypoglycemic activity, process for preparation and use thereof. The process for preparation of  Grifola frondosa  polysaccharide F2 is as follows: The fruit bodies of  Grifola frondosa  were homogenized to a fine powder and extracted with hot water. The mixture was filtered and precipitated with absolute ethanol. The precipitation was obtained. The said precipitation was applied on DEAE Sepharose Fast chromatographic column, equilibrated with Tris-HCl(10 mM, pH=8.0), collecting the efficient eluting peak to obtain the fraction F1; eluted with Tris-HCl(10 mM, pH=8.0) which contains 0.1M NaCl, fraction F2 was obtained; then concentrated under reduced pressure, dialyzed and lyophilized,  Grifola frondosa  polysaccharide F2 was obtained. This isolates a new  Grifola frondosa  polysaccharide F2 with hypoglycemic activity from the fruit bodies of  Grifola frondosa.  The  Grifola frondosa  polysaccharide F2 can be used in manufacturing a drug for treating diabetes. The polysaccharide makes a foundation for developing new anti-diabetes agents.

RELATED APPLICATION

This application claims the benefit of Chinese Patent Application No.201310733480.8, filed on Dec. 25, 2013, the specification of which isincorporated herein with this reference.

TECHNICAL FIELD

This invention relates to a Grifola frondosa polysaccharide F2 withhypoglycemic activity, preparation method and use thereof, belonging tothe field of bio-medicine.

BACKGROUND

Diabetes is a metabolic disorder syndrome characterized byhyperglycemia. The number of diabetes patients around the world has beenincreasing at a speed of 6 percent each year. Between 2010 and 2030, thenumber of adults with diabetes was expected to increase by 69% indeveloping countries and by 20% in developed countries. Diabetesincludes type 1 and type 2 diabetes, with type 2 diabetes accountingmore than 90% of all cases of diabetes. There is currently no knownmethod to conquer diabetes, and the patients' conditions are generallycontrolled and alleviated by orally administered drugs for a long-term.But most of current drugs for diabetes are chemical or biochemicaldrugs, which have much side effects. Based on this critical situation,safer and more effective treatment modalities for diabetes are thereforeneeded to address the increase in prevalence of diabetes.

A polysaccharide with hypoglycemic activity was obtained from fruit bodyof cultivated Grifola frondosa by Xun Ma—α-glucan with a molecularweight of 400-450 KD. In the process of isolating polysaccharides fromfruit body of Grifola frondosa, Kubo obtained a glycoprotein afteradding one volume of ethanol into the hot water extract to generatesuspension and centrifugation. The glycoprotein named X-fraction(polysaccharide:protein=65:35), with a molecular weight of 5×105 D,showed a significant hypoglycemic activity. Structure analysis indicatedthat it was a β-1,6-glucan with α-1,4-branch.

The modern pharmacology studies reveals that blood serum glucose can belowered through increasing insulin levels, regulating the activity ofsome enzymes related to glycometabolism and sequentially acceleratingthe glucose oxidation utilization, and through improving the insulinresistance or inhibition of glucose absorption.

The possible action mechanism of MT-α-glucan from the fruit body ofGrifola frondosa was increasing sensitivity of insulin and improvinginsulin resistance of the surrounding tissue through increasing thenumber of insulin receptor. Water soluble extract FXM from Grifolafrondosa potentially decreased blood serum glucose by improvement theinsulin resistance. Glycoprotein SX from fruit body of Grifola frondosacan improve glucose tolerance and increase the body's sensitivity toinsulin.

It has been found that Grifola frondosa and its active ingredients haveobvious hypoglycemic activity, however, the compositions, structures andmechanisms of most bioactive hypoglycemic ingredients are not fullyunderstood, due to the complex chemical structures. That directlyhinders the process of developing new anti-diabetes agents.

CONTENTS OF THE INVENTION

The object of this invention is to provide a new Grifola frondosapolysaccharide F2 with hypoglycemic activity and preparation methodthereof.

A process for preparation of the above-mentioned Grifola frondosapolysaccharide F2 comprising:

(a) The fruit bodies of Grifola frondosa are homogenized to a finepowder and extracted with hot water. The mixture was filtered andprecipitated with absolute ethanol, and the resulting precipitate of isobtained;(b) The said precipitation is applied on DEAE Sepharose Fast Flowchromatographic column, equilibrated with Tris-HCl(10 mM, pH=8.0),collecting the efficient eluting peak to obtain the fraction F1; elutedwith Tris-HCl(10 mM, pH=8.0) which contains 0.1M NaCl, fraction F2 isobtained; then concentrated under reduced pressure, dialyzed andlyophilized, Grifola frondosa polysaccharide F2 is obtained.

Preferably, the said precipitation in the step (a) is dissolved into hotwater, filtered by a 0.45 μm millipore filter and then dialyzed indialysis bag of 3000 D for 24 h to yield crude polysaccharidessolutions. The concentration of crude polysaccharide in the Grifolafrondosa crude polysaccharides solution is adjusted, and then be used asthe sample of chromatographic separation in the step (b).

The said polysaccharide F2 from Grifola frondosa has a molecular weightof 4.52×10⁵ D. The contents of polysaccharides and protein arerespectively 95.6% and 3.6%. The said polysaccharides is mainlyconsisted of glucose, mannose, xylose, galactose, and arabinose; theamino acids mainly comprises: proline(Pro), serine(Ser), aspariticacid(Asp), lysine(Lys), alanine(Ala), glutamic acid(Glu),threonine(Thr), glycine(Gly), arginine(Arg), leucine(Leu) andvaline(Val). The said polysaccharide F2 is a β-heteropolysaccharidescomposed of uronic acids. Based on the above-mentioned parameters, thereis no known analogous polysaccharide in the existing technology, that isto say, Grifola frondosa polysaccharide F2 of this invention is a newGrifola frondosa polysaccharide.

Pharmacology experiment in vivo showed that when diabetic rats werecontinuously intragastrical administered Grifola frondosa polysaccharideF2 for 7 days, FBG (fasting blood glucose) can be decreasedsignificantly. The mechanism of lower blood glucose levels is mainlythrough improvement insulin resistance.

Accordingly, the second object of the invention is to provide the use ofGrifola frondosa polysaccharide F2 in manufacturing a drug for thetreatment of diabetes, especially for type 2 diabetes.

Another object of the invention is to provide a drug for the treatmentof diabetes, characterized in that the drug comprises Grifola frondosapolysaccharide F2 as active ingredients.

The said diabetes is type 2 diabetes.

This invention isolates a new Grifola frondosa polysaccharide F2 withhypoglycemic activity from the fruit body of Grifola frondosa. It can beused in manufacturing a drug for the treatment of diabetes, especiallyfor type 2 diabetes. The invention makes a foundation for developing newanti-diabetes agents in the future, and actively promotes the study onactive ingredients of natural medicine for treating diabetes.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the elution curve of the precipitation on DEAE SepharoseFast Flow chromatography.

FIG. 2 shows the calibration curve of the standard polysaccharides onGel permeation chromatography.

FIG. 3 shows the HPLC chromatogram of Grifola frondosa polysaccharideF2.

FIG. 4 shows the Infrared spectrum (IR) of Grifola frondosapolysaccharide F2.

FIG. 5 shows the ¹H-NMR spectrum of Grifola frondosa polysaccharide F2.

FIG. 6 shows the ¹³C-NMR spectrum of Grifola frondosa polysaccharide F2.

EXAMPLES

The examples below further illustrate the invention, rather thanlimiting the scope thereof.

Example 1

I. Preparation of Grifola frondosa Polysaccharide F2

The fruit bodies of Grifola frondosa were homogenized to a fine powderand extracted with hot water(5000 mL, 80) for 8 hours. The mixture wasfiltered and the filtrate was concentrated to 1000 mL under reducedpressure, then precipitated with 4 volume of absolute ethanol. Theprecipitation was obtained by centrifugation at a speed of 5000 rpm. Theprecipitation was dissolved into hot water, filtered by a 0.45 μmmillipore filter and dialyzed in dialysis bag of 3000 D for 24 h toyield crude polysaccharides solution. Finally the concentration of thesolution was adjusted to be 10 mg/mL.

The above-mentioned crude polysaccharides solution (100 mL) wasfiltrated and applied on DEAE Sepharose Fast Flow chromatographic column(4.5×30 cm), F1 was obtained by collecting and concentrating thefraction equilibrated with 300 mL Tris-HCl (10 mM, pH=8.0), F2 wasobtained by collecting and concentrating this fraction eluted with 300mL Tris-HCl buffer (10 mM, pH=8.0) which contains 0.1M NaCl. The elutioncurve was shown in FIG. 1.

II. Determination of Purity and Molecular Weight of Grifola frondosaPolysaccharide F2

Grifola frondosa polysaccharide F2 (10 mg) was dissolved into theultrapure water (1 mL) and analyzed by HPLC.

Chromatographic conditions: TSK-GEL G3000SW_(XL) column (300 mm×718 mm);column temperature: 35° C.; mobile phase: 0.05M NaH₂PO₄—Na₂HPO₄ buffer(pH 6.7) containing 0.05% NaN₃; flow rate: 0.5 mL/min; differentialrefractive index detector and constant temperature at 35° C.; injectionvolume: 20 μL.

Establishment GPC calibration curve: 10 mg polysaccharides with Mr of738, 5800, 1.22×10⁴, 2.37×10⁴, 4.80×10⁴, 1.00×10⁵, 1.86×10⁵, 3.80×10⁵,8.53×10⁵ D were used as standards, dissolved into 0.05M NaH₂PO₄—Na₂HPO₄buffer (1 mL, pH 6.7) containing 0.05% NaN₃, filtered by a 0.45 μmmillipore filter and analyzed by GPC. The retention times of thestandard polysaccharides with the known Mr were shown in table 1. GPCcalibration curve was established with elution volume of the standardpolysaccharides as the abscissa and the value of Mr as the verticalcoordinates, see FIG. 2.

Determination of purity and molecular weight of Grifola frondosapolysaccharide F2: 10 mg Grifola frondosa polysaccharide F2 wasdissolved in 0.05M NaH₂PO₄—Na₂HPO₄ buffer (1 mL, pH 6.7) containing0.05% NaN₃, filtered by a 0.45 μm millipore filter and analyzed by GPC.The average molecular weight of Grifola frondosa polysaccharide F2 is4.52×10⁵ D (as shown in FIG. 3), calculated automatically by GPCchromatographic working station.

TABLE 1 The retention times of standard polysacchrides Mr (D) Retentiontime (min) Elution volume (mL) 738 22.6 11.3480 5800 20.1 10.4296 1.22 ×10⁴ 18.3 9.4976 2.37 × 10⁴ 16.3 8.4592 4.80 × 10⁴ 13.8 7.0456 1.00 × 10⁵12.3 6.1120 1.86 × 10⁵ 11.9 5.7824 3.80 × 10⁵ 11.7 5.6615 8.53 × 10⁵11.7 5.6416

III. Physicochemical Property of Grifola frondosa Polysaccharide F2

1. Determination of the Contents of Polysaccharides and Protein

The content of polysaccharides of F2 is 95.6% determined by phenolsulfuric acid method, and that of protein is 3.6% determined by Bradfordmethod.

2. Amino Acid Composition Analysis

HPLC (HP1050, America), consisted of quaternary gradient pump, automaticsampler, column oven and HP1046A fluorescence detector; Full-automatichigh Speed Freezing Centrifuge (2000 rpm, GL20A, Japan).

Standard amino acid, derivatization reagent OPA, FMOC from SIGMA firm;Na₂HPO₄ was of analytical grade; methyl alcohol and acetonitrile were ofHPLC grade.

Chromatographic conditions: Hypersil ODS column (4.0×125 mm, particlesize 5 μm); mobile phase (A): 10 mmol·L⁻¹ pH 7.2 Na₂HPO₄ buffer (PB);mobile phase (B): the mixture of PB, methyl alcohol and acetomitrile(volume percents of them were respectively 50%, 35% and 15%); lineargradient: volume fraction of mobile phase B rise to 40% from 0% linearlyin 0-10 min; volume flow rate: 1.0 mL·min⁻¹; column temperature: 40;determine wavelength: excitation wavelength 340 nm and emissionwavelength 450 nm.

Samples Treatments:

Standard solution: standard amino acids were dissolved into 0.1 mol·L⁻¹HCl solution, and diluted to standard solution, in which theconcentration of each amino acid was 250 nM.

Hydrolysis: putting Grifola frondosa polysaccharide F2 into a hydrolysistube, adding 6 M HCl (10˜15mL) and two drops of newly-distilled phenol.The hydrolysis tube was freezed for 3-5 min in refrigerant, and thenlinked to the exhaust tube of vacuum pump. Vacuum pumping to be closedto 0 Pa and filling with high-purity nitrogen, the treatment wasrepeated three times and the hydrolysis tube was sealed under nitrogen.The sealed hydrolysis tube was put into a constant temperature dryingoven (110±1), after 23 hours, taken out and cooled. Opening and washingthe hydrolysis tube with deionized water, all the hydrolysate wastransferred into a 100 mL volumetric flask and diluted with deionizedwater to volume. The hydrolysate was properly diluted to be detected.

Contrasting to HPLC chromatogram of the standard amino acids, it wasinferred that the amino acids of Grifola frondosa polysaccharide F2mainly comprises: proline(Pro), serine(Ser), asparitic acid(Asp),lysine(Lys), alanine(Ala), glutamic acid(Glu), threonine(Thr),glycine(Gly), arginine(Arg), leucine(Leu) and valine(Val). See table 2.

TABLE 2 Amino acid composition of Grifola frondosa polysaccharide F2Analysis items Results Unit Phenylalanine (Phe) <0.10 g/100 g Alanine(Ala) 0.19 Methionine (Met) <0.10 Proline (Pro) 0.38 Glycine (Gly) 0.15Glutamic acid (Glu) 0.19 Arginine (Arg) 0.11 Lysine (Lys) 0.19 Tyrosine(Tyr) 0.10 Leucine (Leu) 0.10 Serine (Ser) 0.23 Threonine (Thr) 0.17Aspartic acid (Asp) 0.21 Isoleucine (Ile) 0.10 Histidine (His) <0.10Valine (Val) 0.10 Total 2.1

3. Monosaccharide Composition of Grifola frondosa polysaccharide F2analysis by GC-MS

Hydrolysis: Grifola frondosa polysaccharide F2 was dissolved in 2 MH₂SO₄, heated to reflux for 6 hours, cooled and then neutralized withsaturated Ba (OH)₂ to be neutral. The mixed liquid was filtrated and thefiltrate was collected.

Acetylation: the above-mentioned hydrolysate was evaporated to dryness.The residue sample was converted to acetylated derivatives with 70 mghydroxylamine hydrochloride and 5 mL pyridine for 1 hour at 90° C. bywater bath heating. After slightly cooling, 5 mL acetic anhydride wasthen added with heating at 90° C. After 1 hour, 10 mL water was added tobreak the anhydride and the acetylated products was extracted withchloroform. The extract liquor was washed with water and thensubsequently dehydrated with anhydrous Na₂SO₄. The supernate wasconcentrated to 1 mL under nitrogen and analyzed by using GC-MS.

GC-MS operation conditions: SE230 elastic quartz capillary column (15m×012 mm×0133 Lm); the temperature program was set to increase to 280from 100 at a rate of 10/min, then holding for 10 min at 280; carriergas: Helium; column pressure: 70 kPa; split ratio:10:1; solvent delay:2min; electron ionization mode: EI; electron energy:70 eV; quadrupole rodtemperature:150; temperature of ion source:230; voltage of electronmultiplier:2300V; interface temperature of GC-MS:280; Mass scanned range(m/z): 29˜500.

The monosaccharide composition of Grifola frondosa polysaccharide F2 wasanalyzed by GC-MS after hydrolysis and acetylation, as control, thestandard monosaccharides was acetylated simultaneously. According to thetotal ions chromatogram of standard monosaccharides, it can be inferredthat Grifola frondosa polysaccharide F2 is a heteropolysaccharide mainlyconsisted of glucose, mannose, xylose, galactose, arabinose and ribose.The relative content of the monosaccharides were shown in table 3.

TABLE 3 Monosaccharide composition of Grifola frondosa polysaccharide F2Relative content by peak area Sample Monosaccharide normalization NamesComposition (%) Methods Grifola Ribose 1.96 The samples were frondosaArabinose 3.22 analyzed by GC-MS polysaccharide Xylose 8.02 afterhydrolysis and F2 Mannose 16.72 acetylation Glucose 63.74 Galactose 6.75

4. Infrared Spectrum(IR) Analysis of Grifola frondosa polysaccharide F2

1 mg of Grifola frondosa polysaccharide F2 and 100 mg of dried KBr wereporphyrizied and tabletted, the infrared profile of Grifola frondosapolysaccharide F2 was scaned in the range of 400 to 4000 cm⁻¹. Table 4was the analysis of absorption peaks in the infrared spectrum (FIG. 4).According to the infrared spectrum analysis, Grifola frondosapolysaccharide F2 is mainly consisted of pyranoid rings linked byβ-glucosidic bonds.

TABLE 4 Analysis of absorption peaks in the infrared spectrum of Grifolafrondosa polysaccharide F2 Trans- Wave- mit- Num- number tance ber(cm⁻¹) (%) Analysis of the peaks 1 3272.3 15 hydroxy 2 2924.5 16Stretching vibration of C—H 3 1670.5 18 Stretching vibration of C═O 41408 20 Stretching vibration of COO⁻ 5 1064 10 pyranoid rings linked byβ-glucosidic bonds 6 885-900 β-glucosidic bonds

5. NMR Spectrum of Grifola frondosa Polysaccharide F2: ¹H-NMR and¹³C-NMR

The ¹H-NMR spectr of Grifola frondosa polysaccharide F2 was shown inFIG. 5 and the ¹³C-NMR spectrum in FIG. 6.

The signal at 4.395 ppm of ¹H-NMR indicated that Grifola frondosapolysaccharide F2 is mainly consisted of pyranoid rings, in accordancewith the results from infrared spectrum analysis.

In ¹³C-NMR, the chemical shifts of anomeric carton are generally in therange of 90-110 ppm, and the number of glycoside residues depends on thenumber of signal peaks. So Grifola frondosa polysaccharide F2 isconsisted of five glycoside residues. The results of GC-MS showed itcontains six glycoside residues, wherein the fraction of ribose is just1.96%, thus, Grifola frondosa polysaccharide F2 is mainly consisted ofglucose, mannose, xylose, galactose, and arabinose.

According to ¹³C-NMR, the number of glycoside residues and theirrelative contents can be determined by the number of peaks in theresonance area of anomeric carton (90-110 ppm). In general, the chemicalshifts of anomeric carbons from α-glucosides are in the range of 95-101ppm, while that of β-glucosides in the range of 101-105 ppm. Thechemical shifts of Grifola frondosa polysaccharide F2 are respectivelyat 97.94 ppm, 101.30 ppm, 102.26 ppm, 102.92 ppm and 115.47 ppm, it isconsequently considered as a β-heteropolysaccharide.

In addition, the characteristic signals of ¹³C-NMR can determine someglycoside residues and groups, for example, the signals of carboxyls-Cof uronic acids are in the range of 170-180 ppm, and the chemical shiftsof Grifola frondosa polysaccharide F2 are respectively at 171.81 ppm,173.32 ppm, 174.37 ppm, 177.68 ppm and 177.53 ppm, as a consequence,Grifola frondosa polysaccharide F2 contains uronic acids.

In conclusion, Grifola frondosa polysaccharide F2 of the invention has amolecular weight of 4.52×10⁵ D. The contents of polysaccharides andprotein are respectively 95.6% and 3.6%. The said polysaccharides ismainly consisted of glucose, mannose, xylose, galactose, and arabinose;the amino acids mainly comprises: proline(Pro), serine(Ser), aspariticacid(Asp), lysine(Lys), alanine(Ala), glutamic acid(Glu),threonine(Thr), glycine(Gly), arginine(Arg), leucine(Leu) andvaline(Val). The said polysaccharide F2 is a β-pyran heterocyclicpolysaccharide containing uronic acids. Based on the above-mentionedparameters, there is no known analogous polysaccharide in the existingtechnology. Therefore, Grifola frondosa polysaccharide F2 of thisinvention is a new Grifola frondosa polysaccharide.

IV. Pharmacology Experiment

Hypoglycemic experiment of Grifola frondosa polysaccharide F2 on type 2diabeticrats:

(1) Induction of diabetes rats: SD rats at age of 6 weeks (weight:140-160 g), male, were marked and kept in separate cages. They wereadapted for 7 days and then fasted overnight before an intraperitonealinjection of freshly prepared STZ (Sigma, 35 mg/kg body weight [BW],dissolved in citrate buffer, pH 4.5). Then, the rats were fed a high-fatdiet. fter 4 weeks, the rats were fasted for 5 h and fasting serumglucose levels were determined. Rays with fasting serum glucoselevels>11.1 mM were considered to be diabetic and were used in thestudy.(2) Assessment of hypoglycemic activity: Except normal control (NC), thediabetic rats (DM) were randomly divided into three groups: modelcontrol group (MC); low-dose group of Grifola frondosa polysaccharide F2(50 mg/kg BW, Intragastric(ig)); high-dose group of Grifola frondosapolysaccharide F2 (100 mg/kg BW, Intragastric(ig)). The rats of low-doseand high-dose groups were administered different doses of Grifolafrondosa polysaccharide F2 by intragastric infusion, meanwhile, rats ofNC and MC were administered saline. The blood glucose levels weremeasured after continuously giving drugs for 2 weeks. According to table5, FBG were decreased significantly (*P<0.05) afterdiabetic rats werecontinuously administered Grifola frondosa polysaccharide F2 (100 mg/kgBW) for 2 weeks by intragastric infusion. Thus, Grifola frondosapolysaccharide F2 of the invention can be used to manufacturemedicaments for the treatment of diabetes, especially for type 2diabetes.

TABLE 5 Effects of Grifola frondosa polysaccharide F2 on FBG in type 2diabetic rats(ig). Dose Fasting Serum Glucose FSG(mmol/L) Groups(mg/kg/d) 0 w 1 w 2 w NC /  5.68 ± 0.39  5.78 ± 0.61 5.94 ± 0.42 MC /20.65 ± 4.56 25.22 ± 3.94 24.77 ± 4.27  F2  50 21.41 ± 4.29 23.74 ± 2.6723.43 ± 2.98  F2 100 21.41 ± 4.23 23.62 ± 2.97 21.03 ± 3.19* ps:compared to MC: *P < 0.05, ** P < 0.01.

1. A process for preparation of Grifola frondosa polysaccharide F2,comprising: (a) the fruit bodies of Grifola frondosa are homogenized toa fine powder and extracted with hot water; the mixture was filtered andprecipitated with absolute ethanol; the precipitation was obtained. (b)the said precipitation is applied on DEAE Sepharose Fast chromatographiccolumn, equilibrated with Tris-HCl(10 mM, pH=8.0), collecting theefficient eluting peak to obtain the fraction F1; eluted withTris-HCl(10 mM, pH=8.0) which contains 0.1M NaCl, fraction F2 wasobtained; then concentrated under reduced pressure, dialyzed andlyophilized, Grifola frondosa polysaccharide F2 is obtained.
 2. Aprocess for preparation of Grifola frondosa polysaccharide F2 accordingto claim 1, characterized in that the said precipitation in the step (a)is dissolved into hot water, filtered by a 0.45 μm millipore filter anddialyzed in dialysis bag of 3000 D for 24 h to yield crudepolysaccharides; the concentration of crude polysaccharide in theGrifola frondosa crude polysaccharides solution was adjusted, and thenbe used as the sample of chromatographic separation in the step (b). 3.Grifola frondosa polysaccharide F2 prepared by the process forpreparation of Grifola frondosa polysaccharide F2 as claimed in claim 1.4. Use of Grifola frondosa polysaccharide F2 as claimed in claim 3 formanufacturing a drug for the treatment of diabetes.
 5. The use accordingto claim 4, wherein, the said diabetes is type 2 diabetes.
 6. A drug forthe treatment of diabetes, characterized in that the said medicamentcomprises Grifola frondosa polysaccharide F2 as claimed in claim 3 asactive ingredients.
 7. The drug according to claim 6, wherein the saiddiabetes is type 2 diabetes.